Genetic Diversity Of Rice Sheath Blight Fungus And Genes Up-regulated During Early Stage Of Infection To Rice Leaves

Rice sheath blight is a particularly important component of rice disease complex and makes great losses to the yield and quality of rice.In this paper,genetic diversity of rice sheath blight fungus was analyzed among isolates obtained from rice fields of Hubei province,Jiangxi province,Hunan province,Jiangsu province and Guangxi province. Up-regulated genes of strain WH-1 from Wuhan County were screened during the early stage of the infection to rice leaves.1088 isolates were isolated from typical lesions of sheath blight collected from different counties and identified by morphological and molecular techniques.Out of 1088 isolates,1068 belonged to Rhizoctonia solani AG-1â… A,19 were R.oryzae-sativae,and 1 belonged to R.solani AG-7.The results suggested that the anastomosis group 1â… A (teleomorph T.cucumeris) of R.solani is the major group infecting rice.Rich genetic diversity existed among isolates from different fields,even from the same field based on the growth rate,clone morphology,hyphal diameter,karyon type and pathogenicity.77 isolates from one field of Wuhan could be classified into 16 RAPD subgroups at the 0.83 similarity level;and 71 stains isolates from different provinces were classified into 15 RAPD subgroups and the polymorphic bands were not closely related with the geographic location.In addition,3 isolates from one rice field of Guilin,Guangxi province and 1 from Wuhan,Hubei province were different significantly from other isolates.A fungal isolate,GXE4,from Guilin was studied in detail.Compared with normal isolate,isolate GXE4 produced abnormal pigment and less sclerotia,grew more slowly, and was much difficult to produce sclerotia.GXE4 also lost the ability to infect rice when inoculated with hyphal agar.GXE4 was identified to belong to R.solani AG-1â… A based on the result of hyphal fusion,the number of nuclei and the nucleotide sequence of ITS. There exsited two extra linear chromosomal elements(named as 3.6-kb frag-1 and 3.6-kb frag-2) which were proved to be very stable when GXE4 was treated with antibiotic, protoplast regeneration and EB respectively.The DNA of 3.6-kb frags was sensitive to DNaseI but not to RNase,and had no protein or knob-shaped structure in 3'end of 3.6-kb frags.Partial sequence of 3.6-kb flags was cloned and analyzed.There was high similarity between 3.6-kb frag-1 and 3.6-kb frag-2.The DNA of 3.6-kb frag-1 was presumed not to encode any protein longer than 110 amino acids.The DNA of 3.6-kb frag-2 did not have a longer ORF either.The result of genomic DNA southern blot showed that there was a significant hybridization signal with DNA fragments of 3.6-kb frags at 23 kb and 7.0 kb. Based on the results,3.6-kb frags could be defected DNA elements from genome of dsDNA virus or linear plasmid in fungi.Isolate RCOL-1,was obtained from a typical lesion of sheath blight collected from the campus of Huazhong Agricultural University.RCOL-1 grew more slowly than both T. cucumeris and C.oryzae-sativae,and formed abnormal colony on PDA;RCOL-1 also lost the ability to attack rice when inoculated with both hyphal agar and sclerotia.DAPI staining showed that the nuclei pattern in individual cell of RCOL-1 was various,namely some cells contained only one nucleus(1.3%),most contained double nuclei(95.9%), and some cells contained multi-nuclei(2.8%).There existed ITS DNAs of C. oryzae-sativae and T.cucumeris in isolate RCOL-1.Based on the results of PCR amplification using 4 primers specific to T.cucumeris and C.oryzae-sativae,RCOL-1 contained four types ITS DNA,namely ITS DNA of C.oryzae-sativae,ITS DNA of T. cucumeris and two types of chimerical ITS DNA.Two chimerical ITS DNAs were ITS1 of from C.oryzae-sativae and ITS2 of from T.cucumeris,and ITS1 of T.cucumeris and ITS2 of C.oryzae-sativae.The results implied that homologous recombination had occurred at the rDNA region between the two pathogenic fungi.In order to understand the molecular interaction of R.solani AG-1â… A and rice, cDNA library was constructed by suppression subtractive hybridization(SSH) using rice (Oryza sativa) 9311 inoculated with a highly aggressive strain WH-1,and 129 fungal clones expression strengthened significantly during the early stage of infection were obtained.These EST_S were sequenced and analyzed on NCBI non-redundant GenBank database.96 EST_S had significant homology to sequences in the database and 33 had no known homologues in current database.The probable function of the 96 EST_S could be classified into 12 groups according to their putative BLASTX identification.The groups include Metabolism(13%),Protein fate(9%),Protein synthesis(8%),Energy(6%), Signal transduction(5%),Cellular transport(4%),Cell defense(2%),Transcription(1%), Structural(1%),Regulation(1%),Cell cycle(1%) and Unclassified function(49%). Putative proteins of those EST_S included pathogenic signal conduction factor-Mitogen activated protein kinase(MAPK),cyclin-dependent kinase(CDK).These genes may involve in pathogenesis of R.solani and contribute to pathogenesis,and this need further study in the future.